Carbonic anhydrase having increased stability under high temperature conditions

ABSTRACT

The present invention relates to polynucleotide and polypeptide sequences of novel carbonic anhydrase variants having increased stability under high temperature conditions compared to native carbonic anhydrase.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of prior application Ser. No.11/409,487 filed Apr. 20, 2006, which claimed priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 60/673,345 filed on Apr. 21,2005.

FIELD OF THE INVENTION

The present invention relates to polynucleotide and polypeptidesequences of novel carbonic anhydrase variants having increasedstability under high temperature conditions compared to native carbonicanhydrase.

BRIEF DESCRIPTION OF THE PRIOR ART

Carbonic anhydrase (EC 4.2.1.1.) is a globular zinc metalloenzyme ofmolecular weight 30,000 daltons. The enzyme was discovered in 1933 andhas been the subject of intense scientific investigation. Multipleisoforms have been discovered in plant and animal tissues where it isbelieved to facilitate the transport of carbon dioxide. Red blood cellscontain isoenzymes I and II, which are the most active. Carbonicanhydrase II has the highest molecular turnover number of any knownenzyme. One molecule of carbonic anhydrase can hydrate 36,000,000molecules of carbon dioxide in a period of 60 seconds. Physiologically,carbonic anhydrase facilitates the removal of carbon dioxide from themammalian body. The general enzyme reaction is shown below.

CO₂+H₂O

H⁺+HCO₃ ⁻

Human carbonic anhydrase II (CAII) variants have also been the subjectof scientific investigation. Indeed, the functional importance of aconserved hydrophobic face in human carbonic anhydrase II (CAII),including amino acid residues 190-210, was investigated by randommutagenesis.¹

Other CAII variants have been obtained by substituting amino acids ofvarying size at position 65, for instance by changing the amino acid Alafor the amino acid Thr. This modification was done in order toinvestigate the importance of maintaining the active site water networkfor efficient proton transfer.^(2,3)

A library of CAII variants differing in hydrophobic amino acid residuesPhe93, Phe95, and Trp97 was also prepared using cassette mutagenesis,then displayed on filamentous phage, and screened for proteins retaininghigh zinc affinity.⁴

It exists a need in the art for the development of innovative carbonicanhydrase variants harboring advantageous characteristics over nativecarbonic anhydrases, such as exhibiting increased stability under hightemperature conditions.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the residual enzymatic activity of modified carbonicanhydrases II according to preferred embodiments of the inventionfollowing a 2 hour treatment at 55° C.

FIG. 2 shows the residual enzymatic activity of modified carbonicanhydrases II according to preferred embodiments of the inventionfollowing a 2 hour treatment at 60° C.

FIG. 3 shows the residual enzymatic activity of modified carbonicanhydrases II according to preferred embodiments of the inventionfollowing a 2 hour treatment at 62.5° C.

FIG. 4 shows the residual enzymatic activity of modified carbonicanhydrases II according to preferred embodiments of the inventionfollowing a 2 hour treatment at 65° C.

FIG. 5 shows the residual enzymatic activity of modified carbonicanhydrases II according to preferred embodiments of the inventionfollowing a 2 hour treatment at 70° C.

FIG. 6 shows the amino acid sequence of the native unmodified carbonicanhydrase 11 (SEQ ID NO:1).

FIGS. 7 to 16 show the amino acid sequence of modified carbonicanhydrases according to preferred embodiments of the invention (SEQ IDNOS: 2 to 11).

FIGS. 17 to 26 show the nucleotide sequence encoding the carbonicanhydrases of FIGS. 7 to 12 (SEQ ID NOS: 12 to 21).

DETAILED DESCRIPTION OF THE INVENTION

The inventors have surprisingly found that a number of mutations ofhuman carbonic anhydrase II (HCAII), individually or in combination,provide a stabilizing effect on the modified HCAII protein and enableenzymatic activity at higher temperature than normal (i.e higher than25° C.). In this connection, the present invention specifically relatesto the identification of polypeptides and polynucleotide sequencesencoding a modified carbonic anhydrase (CA), preferably of human origin,which have increased stability compared to native CA.

1. DEFINITIONS

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

As used herein, the expression “high temperature conditions” refers totemperature higher than 25° C. and lower than 70° C. Preferably, itrefers to temperature higher than about 37° C., more preferably higherthan about 55° C. and even more preferably higher than about 65° C. By“about”, it is meant that the value of said temperature can vary withina certain range depending on the margin of error of the method orapparatus used to evaluate such temperature. For instance, the margin oferror may range between ±1° C. to ±5° C.

As used herein, the term “polypeptide(s)” refers to any peptide orprotein comprising two or more amino acids joined to each other bypeptide bonds or modified peptide bonds. “Polypeptide(s)” refers to bothshort chains, commonly referred to as peptides, oligopeptides andoligomers, and to longer chains generally referred to as proteins.Polypeptides may contain amino acids other than the 20 gene-encodedamino acids. “Polypeptide(s)” include those modified either by naturalprocesses, such as processing and other post-translationalmodifications, but also by chemical modification techniques. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature, and they arewell known to those of skill in the art. It will be appreciated that thesame type of modification may be present in the same or varying degreeat several sites in a given polypeptide.

A “functional derivative”, as is generally understood and used herein,refers to a protein/peptide/polypeptide sequence that possesses afunctional biological activity that is substantially similar to thebiological activity of the whole protein/peptide/polypeptide sequence.In other words, it refers to a polypeptide of a modified CA of theinvention that substantially retain(s) the capacity of catalyzing thehydration of carbon dioxide. A functional derivative of a modified CAprotein/peptide of the invention may or may not containpost-translational modifications such as covalently linkedcarbohydrates, if such modification is not necessary for the performanceof a specific function. The term “functional derivative” is meant toencompass the “fragments” or “chemical derivatives” of a modified CAprotein/peptide of the invention. As used herein, a protein/peptide issaid to be a “chemical derivative” of a modified CA protein/peptide ofthe invention when it contains additional chemical moieties not normallypart of the protein/peptide, said moieties being added by usingtechniques well known in the art.

By “substantially identical” when referring to a polypeptide, it will beunderstood that the polypeptide of the present invention preferably hasan amino acid sequence having at least 80% identity, or even preferably85% identity, or even more preferably 95% to SEQ ID NOS:1 to 11, orfunctional derivatives thereof.

One can use a program such as the CLUSTAL program to compare amino acidsequences. This program compares amino acid sequences and finds theoptimal alignment by inserting spaces in either sequence as appropriate.It is possible to calculate amino acid identity or homology for anoptimal alignment. A program like BLASTp will align the longest stretchof similar sequences and assign a value to the fit. It is thus possibleto obtain a comparison where several regions of similarity are found,each having a different score. Both types of identity analysis arecontemplated by the present invention.

With respect to protein or polypeptide, the term “isolated polypeptide”or “isolated and purified polypeptide” is sometimes used herein. Thisterm refers primarily to a protein produced by expression of an isolatedand modified polynucleotide molecule contemplated by the invention.Alternatively, this term may refer to a protein which has beensufficiently separated from other proteins with which it would naturallybe associated, so as to exist in “substantially pure” form.

The term “substantially pure” refers to a preparation comprising atleast 50-60% by weight of the modified CA polypeptide of the invention.More preferably, the preparation comprises at least 75% by weight, andmost preferably 90-99% by weight, of the modified CA polypeptide of theinvention.

Purity is measured by methods appropriate for the modified CApolypeptide of the invention (e.g. chromatographic methods, agarose orpolyacrylamide gel electrophoresis, HPLC analysis, and the like).

As used herein, the term “polynucleotide(s)” generally refers to anypolyribonucleotide or poly-deoxyribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. This definition includes, withoutlimitation, single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions or single-, double- andtriple-stranded regions, cDNA, single- and double-stranded RNA, and RNAthat is a mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded, or triple-stranded regions, or a mixture ofsingle- and double-stranded regions. The term “polynucleotide(s)” alsoembraces short nucleotides or fragments, often referred to as“oligonucleotides”, that due to mutagenesis are not 100% identical butnevertheless code for the same amino acid sequence.

By “substantially identical” when referring to a polynucleotide, it willbe understood that the polynucleotide of the invention has a nucleicacid sequence which is at least 65% identical, more particularly 80%identical and even more particularly 95% identical to any one of SEQ IDNO 12 to 21 or functional fragments thereof.

A “functional fragment”, as is generally understood and used herein,refers to a nucleic acid sequence that encodes for a functionalbiological activity of protein that is substantially similar to thebiological activity of protein coding of the whole nucleic acidsequence. In other words, it refers to a nucleic acid or fragment(s)thereof that substantially retains the capacity of encoding a carbonicanhydrase polypeptide of the invention.

The term “fragment”, as used herein, refers to a polynucleotide sequence(e.g., cDNA) which is an isolated portion of the subject nucleic acidconstructed artificially (e.g., by chemical synthesis) or by cleaving anatural product into multiple pieces, using restriction endonucleases ormechanical shearing, or a portion of a nucleic acid synthesized by PCR,DNA polymerase or any other polymerizing technique well known in theart, or expressed in a host cell by recombinant nucleic acid technologywell known to one of skill in the art.

With reference to polynucleotides of the invention, the term “isolatedpolynucleotide” is sometimes used. This term, when applied to DNA,refers to a DNA molecule that is separated from sequences with which itis immediately contiguous (in the 5′ and 3′ directions) in the naturallyoccurring genome of the organism from which it was derived. For example,the “isolated polynucleotide” may comprise a DNA molecule inserted intoa vector, such as a plasmid or virus vector, or integrated into thegenomic DNA of a procaryote or eucaryote. An “isolated polynucleotidemolecule” may also comprise a cDNA molecule.

Amino acid or nucleotide sequence “identity” and “similarity” aredetermined from an optimal global alignment between the two sequencesbeing compared. An optimal global alignment is achieved using, forexample, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol. Biol. 48:443-453). “Identity” means that an amino acid ornucleotide at a particular position in a first polypeptide orpolynucleotide is identical to a corresponding amino acid or nucleotidein a second polypeptide or polynucleotide that is in an optimal globalalignment with the first polypeptide or polynucleotide. In contrast toidentity, “similarity” encompasses amino acids that are conservativesubstitutions. A “conservative” substitution is any substitution thathas a positive score in the blosum62 substitution matrix (Hentikoff andHentikoff, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919). By thestatement “sequence A is n % similar to sequence B”, it is meant that n% of the positions of an optimal global alignment between sequences Aand B consists of identical residues or nucleotides and conservativesubstitutions. By the statement “sequence A is n % identical to sequenceB”, it is meant that n % of the positions of an optimal global alignmentbetween sequences A and B consists of identical residues or nucleotides.

2. MODIFIED CA POLYNUCLEOTIDES AND POLYPEPTIDES OF THE INVENTION

In a first embodiment, the present invention concerns a modifiedcarbonic anhydrase polypeptide having increased stability under hightemperature conditions compared to unmodified carbonic anhydrase, i.e. amodified CA that satisfactory retains enzymatic activity at atemperature higher than suitable for use with native CA (for instancehigher than about 25° C.). As used herein, the term “modified CA” refersto forms of CA that differ structurally from unmodified CA. Inparticular, the modified CA protein of the invention comprise an aminoacid sequence substantially identical to SEQ ID NO 1 and wherein themodified CA comprises at least one amino acid substitution at aposition, or at an equivalent position, corresponding to position 65,93, 100, 136, 153, 198, 223, 239 and 247 of SEQ ID NO 1. In thisconnection, the term “equivalent position” denotes a position which, onthe basis of an alignment of the amino acid sequence of the parentcarbonic anhydrase in question with the “reference” carbonic anhydraseamino acid sequence in question (for example the sequence shown in SEQID No. 1) so as to achieve juxtapositioning of amino acidresidues/regions which are common to both, corresponds most closely to aparticular position in the reference sequence in question.

The substituted amino acid is selected such that and as previouslymentioned, the modified CA retains catalytic activity (i.e. theinterconversion of CO₂ with HCO₃— and H+) and exhibits increasedstability compared to unmodified CA. The term “substituted amino acid”is intended to include natural amino acids and non-natural amino acids.Non-natural amino acids include amino acid derivatives, analogues andmimetics. As used herein, a “derivative” of an amino acid refers to aform of the amino acid in which one or more reactive groups on thecompound have been derivatized with a substituent group. As used hereinan “analogue” of an amino acid refers to a compound that retainschemical structures of the amino acid necessary for functional activityof the amino acid yet also contains certain chemical structures thatdiffer from the amino acid. As used herein, a “mimetic” of an amino acidrefers to a compound in that mimics the chemical conformation of theamino acid.

Preferred amino acid substitutions consist of Ala65Thr, Phe93Leu,Leu100His, Gln136Tyr, Gln136His, Lys153Leu, Leu198Met, Leu223Ser,Leu239Pro or Ala247Thr. More particularly, the modified CA of theinvention comprises an amino acid sequence substantially identical to asequence selected from the group consisting of SEQ ID NOS: 2 to 11 orfunctional derivatives thereof.

It will be understood that while the modified CA of the invention maycomprises only one amino acid substitution at a position, or at anequivalent position, corresponding to position 65, 93, 100, 136, 153,198, 223, 239 and 247 of SEQ ID NO 1, it may be advantageous to providea modified CA which comprises a combination of any of the amino acidsubstitution mentioned above. In other words, the present invention alsoadvantageously concerns a modified CA protein that comprises acombination of two (2×), three (3×), four (4×), five (5×), six (6×),seven (7×), eight (8×) or of nine (9×) of the amino acid substitutionsmentioned above. Preferred combinations contemplated by the presentinvention are those shown in Table 1.

In another embodiment, the present invention concerns an isolatedpolynucleotide encoding a modified CA polypeptide of the invention.Preferably, the isolated polynucleotide of the invention comprises anucleotide sequence substantially identical to a sequence selected fromthe group consisting of SEQ ID NOs: 12 to 21 and functional fragmentsthereof.

2. VECTOR

In another embodiment, the invention is further directed to a vector(e.g. cloning or expression vector) comprising a polynucleotide sequenceof the invention.

As used herein, the term “vector” refers to a polynucleotide constructdesigned for transduction/transfection of one or more cell types.Vectors may be, for example, “cloning vectors” which are designed forisolation, propagation and replication of inserted nucleotides,“expression vectors” which are designed for transcription of anucleotide sequence in a host cell, or a “viral vector” which isdesigned to result in the production of a recombinant virus orvirus-like particle, or “shuttle vectors”, which comprise the attributesof more than one type of vector.

A number of vectors suitable for stable transfection of cells andbacteria are available to the public (e.g. plasmids, adenoviruses,baculoviruses, yeast baculoviruses, plant viruses, adeno-associatedviruses, retroviruses, Herpes Simplex Viruses, Alphaviruses,Lentiviruses), as are methods for constructing such cell lines. It willbe understood that the present invention encompasses any type of vectorcomprising any of the polynucleotide molecule of the invention.

3. CELLS

In a further embodiment, the invention is also directed to a host, suchas a genetically modified cell, comprising any of the polynucleotidesequence according to the invention and more preferably, a host capableof expressing the polypeptide encoded by this polynucleotide. Even morepreferably, the present invention is concerned with a host cell thatincorporates an expression vector or a recombinant viral vector asdefined herein below.

The host cell may be any type of cell (a transiently-transfectedmammalian cell line, an isolated primary cell, or insect cell, yeast(Saccharomyces cerevisiae or Pichia pastoris), plant cell,microorganism, or a bacterium (such as E. coli).

4. USES OF MODIFIED CA

The modified CA proteins of the invention retain the catalytic activityof unmodified CA. Accordingly, the modified CA proteins are useful forcatalysing CO₂. Moreover, since a modified CA protein of the inventionhas increased stability under high temperature compared to unmodifiedCA, a particular amount of this modified CA protein exhibits greatercatalytic activity over time than an equal amount of unmodified CA.

The modified CA proteins of the invention can also be used in processessuch as those described in the following Canadian references: 2.291.785;2.329.113 and 2.393.016.

In addition to the foregoing uses, the modified CA proteins of theinvention, because of their enhanced stability, are particularlywell-suited for removing CO₂ from a CO₂ containing effluent, such as agaseous or liquid effluent, and more particularly for transforming CO₂in high temperature gaseous effluent such as industrial gaseouseffluents.

EXAMPLES

The present invention will be more readily understood by referring tothe following examples. These examples are illustrative of the widerange of applicability of the present invention and are not intended tolimit their scope. Modifications and variations can be made thereinwithout departing from the spirit and scope of the invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice for testing of the present invention,the preferred methods and materials are described.

Example 1 Cloning of Wild-Type Human Carbonic Anhydrase II (HCAII)

Human lymphocytes were isolated from 8 ml of blood, which 7 ml of PBSbuffer (phosphate buffer 20 mM, pH 7.4, 150 mM NaCl) was added. Thisblood/PBS solution was then poured onto a 10 ml Ficoll cushion(Ficoll-Paque, Pharmacia) and centrifuged during 45 minutes at 1900rpm/22° C. (Megafuge 1.0R, Heraeus instruments). The lymphocytes layerwas washed in PBS and then centrifuged at 1900 rpm during 10 minutes toobtain a pellet. The pellet was then resuspendent in TRI-Reagent(Molecular Research inc.) was then added to the pellet for RNAisolation. mRNAs were used to form a cDNA by the SuperScriptII reversetranscriptase (Gibco/BRL) according to the manufacturer's instruction.The oligonucleotide used for the reverse transcription was 5′TTTTTTTTTTTTNV 3′.

HCAII cDNA was amplified by PCR with the following specificoligonucleotides:

5′ ATGTCCCATCACTGGGGGTAC 3′ 5′ TTATTTGAAGGAAGCTTTGATTTGC 3′.

The amplification was carried out in a thermocycler (Applied Biosystemsmodel 9700) according to the following program: denaturation at 94° C.×2min followed by 30 amplification cycles: 94° C.×30 sec, 48° C.×45 sec,72° C.×60 sec and a final extension of the products at 72° C.×7 min. ThePCR product was TA-cloned in the pCR2.1 vector (Invitrogene) accordingto the manufacturer's instructions. The ligation products have beenintroduced into competent E. coli DH5α (Gibco/BRL) and the transformantswere selected on LB-ampicillin agar (100 μg/mL of ampicillin; 80 mg/LX-gal and 0.2 mM IPTG).

The HCAII sequence integrity has been confirmed by direct and reversesequencing. The coding sequence has been cloned in the expression vectorpET28a(+) (NOVAGEN) to provide the pET28a+HCAII vector. Competent E.coli BL21 λDE3 pLysS have been transformed with the ligation productsand the transformants were selected on LB agar with kanamycin (30μg/mL). HCAII synthesis by transformed E. coliλDE3 pLysS in LB broth hasbeen initiated at OD₆₀₀ ˜0.6 by adding IPTG (0.4 mM) and ZnSO₄ (0.5 mM).Synthesis was maintained at 37° C. during 4h00 with an agitation of 250rpm. Recombinant carbonic anhydrase production was confirmed bySDS-PAGE.

Example 2 Preparation of Modified Carbonic Anhydrases by RandomMutagenesis by PCR

Mutagenic amplification of the HCAII coding sequence was preformed withthe pET28a+HCAII vector. The following oligonucleotides were used:

5′ CAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATAT ACCGTGGTAATG 3′5′ GGCTTGCCTGGTGCTCGAGTCATTA 3′

These primers hybridize to the extremities of the HCAII coding sequence.The first primer hybridises upstream to the HCAII coding sequence to thestart codon (ATG) and contains a XbaI restriction site. The secondprimer hydridises downstream to the HCAII coding sequence to the stopcodon (TAA) and contains a XhoI restriction site. The restriction sitesXbaI and XhoI are useful during the cloning steps of the amplicons, asbetter detailed hereinafter.

The amplification reaction mixture consist of TRIS-HCl pH 8.3 at 10 mM(Sigma), KCl (Sigma) 50 mM, MnCl₂ (Sigma) 0.15 mM, gelatine 0.01%(Biorad), ATP 2 mM, GTP 2 mM, TTP 10 mM, CTP 10 mM (dNTP Invitrogene),primers 0.5 mM, Taq DNA polymerase 0.5 Units (Promega), plasmid 4 ng in50 μL. The amplification was performed under the following program:first cycle: 95° C.×2 min., 55° C.×30 sec and 72° C.×30 sec and 14cycles of 95° C.×30 sec., 55° C.×30 sec and 72° C.×30 sec.

The PCR products and the pET28a(+) vector were enzymatically cut by XbaIet XhoI restriction enzymes (Roche), and the resulting fragments werepurified with the QIAquick DNA extraction gel kit (Qiagen) and ligatedwith T4 DNA ligase (Invitrogen). The recombinant molecules were thenintroduced by transformation in competent E. coli BL21 λDE3 pLysS.Transformed bacteria were grown on 20 cm×20 cm petri dishes containingLB agar with kanamycin (30 μg/mL) and chloramphenicol (34 μg/mL).Bacteria were incubated at 37° C. overnight. The petri dishes harbouringapproximatively 2000-3000 UFC were then photographed.

Example 3 Mutant CA selection Arboring Increased Carbonic AnhydraseStability Colony Transfert

The selection method of thermostabilized carbonic anhydrase wasdeveloped according to the method developed by Krebs and Fierke.Following transformation, E. coli colonies were transferred onto a 20cm×20 cm nitrocellulose filter (Hybon-C Extra; Amersham). Thenitrocellulose filter was then disposed with the colonies facing up,onto a second agar containing IPTG (2 mM) and ZnSO₄ (0.5 mM) forinitiating synthesis of carbonic anhydrase recombinants. The inductionwas maintained during four hours at 37° C. The membrane was then placedat −80° C. for at least 30 minutes. The original agar was kept at 4° C.and the membrane was used for the enzymatic assays as describedhereinafter.

Membrane Preparation for Enzymatic Assays

The membrane is defrosted at room temperature for 10 minutes. Themembrane frost-defrost process induces cellular breakings during thedefrosting period which support the release of the cellular contents,such as the lysosyme coded by the pLysS plasmid. This lysosyme causesthe lysis of the bacteria and thus, the release of the cytoplasmiccontents of the bacteria. The nitrocellulose strongly maintains thereleased proteins by electrostatic interactions, such as the HCAII(Human Carbonic Anhydrase II) locally over-expressed by the transferredcolony. The unoccupied sites on the membrane are blocked using a 100 mMTRIS (Sigma)/10 mM NaCl (Sigma) pH 8.0/5 w.v % powder skimmed milk(Nestle) solution, during the first 10 minutes without any agitation andthen 50 minutes using a 50 RPM agitation at room temperature. Then themembrane is washed five times using a 100 mM TRIS/10 mM NaCl pH 8.0solution (1×15 minutes and 4×5 minutes). After the second 5-minutewashing, the fragments of the colonies which have remained linked areremoved softly using a gloved finger. After the last washing, a 25 mLpipette is rolled upon the membrane surface using an adequate pressurein order to expel a maximum quantity of buffer from the membrane.Finally, the membrane is soaked in a 25 mM TAPS (Sigma)/100 mM Na₂SO₄(Fisher) pH 8.4 buffer compatible with enzymatic assays.

Example 4 CA Activity Detection

The selection method uses the hydration activity of CO₂ from the HCAII

$\begin{matrix}{{{Residual}\mspace{14mu} {activity}\mspace{14mu} (\%)} = {\left( \frac{{Activity}\mspace{14mu} {after}\mspace{14mu} {treatment}}{{Activity}\mspace{14mu} {before}\mspace{14mu} {treatment}} \right) \times 100.}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

The hydration reaction of CO₂ releases a proton, leading to a localacidification of the membrane, more specifically where an active HCAIIis located. The membrane is coloured using the following buffer: 25 mMTAPS/100 mM Na₂SO₄ pH 8.4/10 mM purple m-cresol (Fisher). Then, themembrane is placed in a container designed specifically for thispurpose. This container is translucent which allows for the observationof the enzymatic reaction, and contains pure CO₂. The CO₂ hydrationreaction leads to a local quick color change on the membrane from purpleto yellow, where the HCAII, which is able to hydrate the CO₂, islocated. After a first control assay, the membrane is subjected to a 15min. thermal treatment at 53° C. by soaking it in a 53° C. pre-heatedbuffer 25 mM TAPS/100 mM Na₂SO₄ pH 8.4. This is the minimal treatmentnecessary to completely eliminate the signal created by the native HCAIIand allows the activity of the improved mutant enzymes to behighlighted. A second developing process of the membrane as describedabove is performed after the thermal process, and the permanent signalsfrom stabilized HCAII are located. For every enzymatic activity assayson membranes, a numeric camera is used in order to record all the dataand facilitate their later analysis.

Example 5 Identification of Thermostabilized CA Mutants

The numeric signals were analyzed using Adobe Photoshop® software tosuperimpose the image of the above mentioned membrane with thecorresponding Petri-dish culture and identify the clones responsible forthe synthesis of a stabilized mutant HACII.

The identified clones are regrown on nutrient agar in order to validatethis result. The plasmidic DNA of the clones producing stabilized HCAIIis purified and the DNA sequence coding these enzymes is sequenced. (SeeFIGS. 17 to 26)

Example 6 Purification of Thermostabilized CA Mutants for EnzymaticAssay

The cloned recombinant CA was purified according to the followingmethod.

The bacterial pellets were lysed with 18 mL of Lysis buffer (50 mMacetate pH 6.2 with 4 μg/mL of DNAse).

The lysed pellet solutions were centrifuged at 32 000 g during 15minutes. The pH of the supernatant was adjusted to 6.2. The supernatantswere then filtered (0.2 μm). The filtered supernatants were applied on 2mL column of cathionic Unosphere S resin (BioRad). A first wash with 4mL of lysis buffer followed by a second wash of 2 mL of lysis bufferwith 10 mM NaCl were performed. An elution with 15 mL of lysis bufferwith 75 mM NaCl was further performed.

The obtained fractions were applied onto a SDS-Page gel for theidentification of fractions containing the pure CA. Then, thesefractions were pooled.

The concentration of purified CA was measured at 280 nm with a molarabsortion coefficient (epsilon)=5.4×10⁴ M⁻¹ cm⁻¹.

Example 7 CA Enzymatic Assay with Purified Modified CA of the Invention

0.25 mL PCR (Sarstedt) containing 250 μl of purified CA (10 μM) wereincubated for 2 hours at different temperatures (55° C., 60° C., 62.5°C., 65° C. and 70° C.) in a heat bath.

The tubes were then cooled rapidly on ice for stopping the thermaldenaturation. The tubes were then centrifuged (quick spin) at 13 000 RPMto obtain a pellet. The thermostability of the modified CA of theinvention was determined by measuring the residual esterase activityaccording to the above described equation 1.

The esterase activity was measured in buffer TRIS (pH 8,0; 0.1 ionicforce; 1% acetone; 0.5 mM pNPA at a temperature of 25° C.). The enzymeconcentration used was 0.1 μM (40 uL of the treated solution). Theesterase activity was measured by spectrophotometer at 348 nm. Theresults are shown in FIGS. 1 to 5.

REFERENCES

All publications, patents and patent applications cited herein arehereby incorporated by reference in their entirety.

-   1. Krebs, J., Fierke, C., 1993, J. of Biological Chemistry, Vol.    268, p. 948.-   2. Jackman, J. E., Merz, K. M., Jr., & Fierke, C. A. (1996)    Biochemistry 35, 16421-   3. Scolnick L R, Christianson D W., 1996, Biochemistry, Vol 35, No.    51 p. 16429-   4. Hunt. J A, Fierke C A., 1997, J. of Biological Chemistry, Vol.    272, No. 33, p. 20364

TABLE 1 Results of the residual enzymatic activity of modified carbonicanhydrase following a 2 hour treatment under high temperatureconditions. ID. Mutations 55 C. 60 C. 62.5 65 C. 70 C. 0 HCAIIwt 12.6%0.0% 0.0% 0.0% 0.0% 1 A65T 67.9% 0.0% 0.0% 0.0% 0.0% 2 F93L 62.7% 0.0%0.0% 0.0% 0.0% 3 L100H 60.7% 0.0% 0.0% 0.0% 0.0% 4 Q136H 51.5% 0.0% 0.0%0.0% 0.0% 5 Q136Y 62.7% 0.0% 0.0% 0.0% 0.0% 6 K153L 55.0% 0.0% 0.0% 0.0%0.0% 7 L198M 59.7% 0.0% 0.0% 0.0% 0.0% 8 L223S 83.0% 0.0% 0.0% 0.0% 0.0%9 L239P 70.8% 0.0% 0.0% 0.0% 0.0% 10 A247T 84.4% 0.0% 0.0% 0.0% 0.0% 1 +3 2X 100.0% 85.0% 15.3% 0.0% 0.0% 1 + 3 + 9 3X 100.0% 95.0% 61.0% 0.0%0.0% 1 + 3 + 6 + 9 4X 100.0% 100.0% 68.2% 6.6% 0.0% 1 + 3 + 6 + 8 + 9 5X100.0% 100.0% 92.4% 76.4% 0.0% 1 + 3 + 6 + 8 + 9 + 10 6X 100.0% 100.0%100.0% 88.4% 0.0%

1-20. (canceled)
 21. A polynucleotide encoding a modified carbonicanhydrase polypeptide having increased stability under high temperatureconditions compared to unmodified native carbonic anhydrase, saidmodified carbonic anhydrase comprising an amino acid sequencesubstantially identical to SEQ ID NO 1 and functional derivativethereof, and wherein said modified carbonic anhydrase comprises at leastone amino acid substitution at a position, or at an equivalent position,corresponding to position 65, 93, 100, 136, 153, 198, 223, 239 and 247of SEQ ID NO
 1. 22. The polynucleotide of claim 21, wherein said aminoacid substitution consists of Ala65Thr.
 23. The polynucleotide of claim21, wherein said amino acid substitution consists of Phe93Leu.
 24. Thepolynucleotide of claim 21, wherein said amino acid substitutionconsists of Leu100His.
 25. The polynucleotide of claim 21, wherein saidamino acid substitution consists of Gln136Tyr.
 26. The polynucleotide ofclaim 21, wherein said amino acid substitution consists of Gln136His.27. The polynucleotide of claim 21, wherein said amino acid substitutionconsists of Lys153Leu.
 28. The polynucleotide of claim 21, wherein saidamino acid substitution consists of Leu198Met.
 29. The polynucleotide ofclaim 21, wherein said amino acid substitution consists of Leu223Ser.30. The polynucleotide of claim 21, wherein said amino acid substitutionconsists of Leu239Pro.
 31. The polynucleotide of claim 21, wherein saidamino acid substitution consists of Ala247Thr.
 32. The polynucleotide ofclaim 21, wherein the polypeptide comprises an amino acid sequencesubstantially identical to a sequence selected from the group consistingof SEQ ID NOS 2 to 11 or functional derivative thereof.
 33. Thepolynucleotide of claim 21, comprising a nucleic acid sequence which issubstantially identical to any one of SEQ ID NOS 12 to 21 or functionalfragments thereof.
 34. An expression or cloning vector comprising apolynucleotide as defined in claim
 21. 35. A polynucleotide encoding amodified carbonic anhydrase polypeptide having increased thermalstability compared to unmodified native carbonic anhydrase, saidpolynucleotide comprising a nucleic acid sequence which is substantiallyidentical to SEQ ID NOS: 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21.